Glial cells are critical regulators of nervous system function and play a central role in the response of the brain and spinal cord to injury. We and others have shown that glial cells are capable of multiple distinct patterns of intercellular calcium signaling, including bidirectional calcium signaling with neurons. The long range goal of this study is an increased understanding of these glial-glial and glial-neuronal Ca2+ signaling mechanisms and their roles in both the normal physiology of the central nervous system and its response to injury. This study proposes to test the following specific hypotheses: 1) Glial intercellular calcium waves are mediated by both gap junctional coupling and the release of an identifiable extracellular messenger. 2) Glial intercellular calcium waves are associated with propagated changes in glial membrane potential. 3) Bi- directional glial-neuronal Ca2+ signaling occurs both via gap junctional coupling and via release of extracellular messengers. 4) Glial intercellular Ca2+ signaling mediates the response of glia to the conditions of traumatic, hypoxic, and excitoxic injury, including the development of cellular changes typical of gliosis. 5) Bi-directional glial-neuronal Ca2+ signaling mediates the response of both glia and neurons to traumatic, hypoxic, and excitotoxic injury. We will use primary cultures of glia and neurons from the mouse brain cortex, as well as cultures of neuronal, astrocyte, and oligodendrocyte cell lines as models to study spontaneous and injury-induced signaling mechanisms. We will quantify cellular activity using fluorescence video imaging techniques, patch clamp techniques, time-lapse phase contrast video recordings, and immunofluorescence staining. We will correlate intercellular Ca2+ signaling with changes in membrane potential, cell volume, cell morphology, cell viability, and expression of glial fibrillary acidic protein in astrocytes. We will also investigate the effects of pharmacological agents on Ca2+ signaling and correlated cellular parameters, with the goal of identifying pharmacological approaches to the modification of the glial and neuronal response to injury. Therapeutic intervention based on modulation of glial cell responses may significantly alter functional recovery following traumatic or hypoxic-ischemic injury to the brain and spinal cord. An increased understanding of glial cell signaling mechanisms is crucial to the development of these new therapeutic approaches.